Corresponding author: Andrea Matern (
Academic editor: D.J. Kotze
Only very few cases have documented that an increase in connectivity after a period of fragmentation in ecological time has had an effect on the distribution, genetic structure and morphology of stenotopic species. In this study we present an example of clinal variability in a woodland ground beetle as a result of changes in the connectivity of a landscape during the last two centuries. The study area hosts both the nominate form
The history of a landscape has a tremendous effect on both the species composition of communities and assemblages on the one hand and the genetic variability of species on the other. This is especially true for woodlands, which have become highly fragmented since the Middle Ages in large areas of north-western Europe including Britain, southern Scandinavia, Belgium, the Netherlands and the lowlands of northern Germany (
At the genetic level some studies have shown a strong differentiation between remnants of stenotopic woodland species in ancient woodlands that were at least in former times isolated from one another (e.g.
In this study we present an example of clinal variability as the result of an increase in the connectivity of a landscape during the last two centuries. We selected a network of woodlands in north-western Germany that stretches between the only two ancient woodland remnants in the region, and studied the genetic variability and differentiation of the woodland specialist
The study area is located northwest of Osnabrück in the morainic hill country between the convent of Börstel and Bramsche (
The abdomina of
Allele frequencies, observed heterozygosity (
Data were investigated for the occurrence of clinal variation by spatial autocorrelation analysis implemented in SGS ver. 1.0 d (Spatial Genetics Software,
After material had been taken for allozyme analysis, the animals were placed in Scheerpeltz solution (70% ethanol, 5–10% acetic acid, 15–20% aqua dest.). Male genitalia were prepared and mounted on cards. The remains of the exoskeletons were pinned to dry and deposited in the entomological collection of Thorsten Assmann, Bleckede (to be donated to the Zoological State Collection, Munich).
Two morphological properties that are relevant for taxonomic distinction between both forms of
Aedeagus tip of
As a second morphological character, we used elytron sculpture to classify individuals into the following categories: Class “0” for animals with typically smooth elytra as in the nominate form of
A total of 21 alleles were scored at three loci across the 12 populations studied. The number of alleles detected at each locus ranged from five (MPI) to nine (GPI). Allele frequencies, expected and observed heterozygosities and FIS values are shown in Table 1. No significant deviations from Hardy-Weinberg equilibrium were observed for any of the populations or loci after correcting for multiple tests (nominal level of p = 0.05). There seems to be a tendency of populations to display a positive FIS, i.e. a deficit of heterozygotes. However, after correction for multiple testing by Fstat (nominal level of p = 0.05, 720 randomizations), no significant deficit or excess of heterozygotes were found. No significant linkage disequilibrium was found, thus the studied loci can be interpreted as independent markers.
The overall FST value was 0.160 and ranged from 0.127 (GPI) to 0.201 (EST-X). Pairwise population differentiation in FST between the 78 pairs in our study ranged between 0.011 and 0.501, with a significant differentiation for 54 population pairs after standard Bonferroni corrections (
Spatial genetic structure analysis revealed gradients in allele frequencies in the EST-X locus, in the MPI locus, and in the whole sample (
Correlogram showing the result of spatial autocorrelation analysis at three allozyme loci. Genetic distances D (
The vast majority of beetles in the northernmost populations 1 to 4 and population 9 showed the typical smooth elytra of the nominate form, while more than half of the beetles in each of the three southernmost populations (10 – 12) showed the elytron sculpture typical of
Both the maximum width and the ratio of maximum width to minimum width show significant differences among population medians (AedMax: H(11, N = 220) = 71.157 p < 0.001; AedMax/AedMin: H(11, N = 219) = 22.737 p = 0.019). The pattern of AedMax is very similar to that of the elytron properties. The maximum width of the genital tip is generally highest in populations adjacent to the northern ancient woodland Börsteler Wald, which is characteristic of
Maximum width of the aedeagus tip
The gradients found in
Numerous studies have dealt with habitat fragmentation at different levels from populations to whole communities. In general, habitat fragmentation has led to genetic differentiation and extinction processes at the population level and is discussed as one of the driving forces for the loss of species worldwide (
Our study provides a case of colonization as a result of increased connectivity by means of hedges and afforestation – even though this may not have been the major aim of anthropogenic landscape changes. This is an encouraging example for nature conservation, which generally aims to purposefully reconnect fragmented landscapes. The gradients detected for
Elytral sculpture, aedeagus tips and allozymes show that strongly differentiated populations of
Elytral sculpture is especially well suited for an unambiguous distinction between both forms, whereas the width of the aedeagus shows significantly differentiated groups, but is a more or less continuously or clinally varying property in the populations. We found many hybrid populations with regards to both properties. Also
Identifying typical or exclusive alleles for either
Diversity of allelic variation.
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N | 70 | 64 | 68 | 46 | 54 | 92 | 22 | 38 | 58 | 48 | 52 | 6 |
1 | 0 | 0 | 0.015 | 0.022 | 0.259 | 0.413 | 0.091 | 0.211 | 0.052 | 0.042 | 0.173 | 0 |
2 | 0.714 | 0.875 | 0.824 | 0.783 | 0.574 | 0.337 | 0.182 | 0.342 | 0.707 | 0.146 | 0.154 | 0.333 |
3 | 0.229 | 0 | 0.015 | 0.043 | 0.074 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
4 | 0.057 | 0.125 | 0.147 | 0.087 | 0.056 | 0.207 | 0.682 | 0.395 | 0.103 | 0.354 | 0.308 | 0.5 |
5 | 0 | 0 | 0 | 0 | 0 | 0.011 | 0 | 0 | 0.086 | 0.25 | 0 | 0 |
6 | 0 | 0 | 0 | 0.065 | 0.037 | 0.033 | 0.045 | 0.053 | 0.052 | 0.208 | 0.327 | 0.167 |
7 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.038 | 0 |
HO | 0.257 | 0.25 | 0.265 | 0.348 | 0.37 | 0.609 | 0.545 | 0.684 | 0.448 | 0.5 | 0.462 | 0.333 |
HE | 0.441 | 0.222 | 0.304 | 0.382 | 0.604 | 0.679 | 0.515 | 0.698 | 0.485 | 0.762 | 0.758 | 0.733 |
FIS | 0.420 | -0.127 | 0.132 | 0.090 | 0.392 | 0.105 | -0.062 | 0.021 | 0.077 | 0.348 | 0.396 | 0.600 |
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N | 76 | 96 | 86 | 80 | 72 | 114 | 28 | 48 | 56 | 70 | 84 | 100 |
1 | 0 | 0 | 0 | 0 | 0 | 0.009 | 0 | 0 | 0 | 0 | 0.024 | 0 |
2 | 0 | 0 | 0.012 | 0 | 0.028 | 0.018 | 0 | 0.083 | 0 | 0.043 | 0.06 | 0.07 |
3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.071 | 0 | 0 |
4 | 0.263 | 0.135 | 0.093 | 0.225 | 0.347 | 0.377 | 0.536 | 0.208 | 0.304 | 0.257 | 0.417 | 0.42 |
5 | 0 | 0 | 0.058 | 0.05 | 0.097 | 0.096 | 0 | 0.063 | 0.071 | 0 | 0.012 | 0.04 |
6 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.01 |
7 | 0.697 | 0.813 | 0.767 | 0.65 | 0.25 | 0.254 | 0.179 | 0.25 | 0.196 | 0.414 | 0.286 | 0.21 |
8 | 0.013 | 0.01 | 0.047 | 0.075 | 0.278 | 0.246 | 0.286 | 0.396 | 0.357 | 0.214 | 0.202 | 0.25 |
9 | 0.026 | 0.042 | 0.023 | 0 | 0 | 0 | 0 | 0 | 0.071 | 0 | 0 | 0 |
HO | 0.421 | 0.375 | 0.372 | 0.375 | 0.611 | 0.649 | 0.5 | 0.667 | 0.607 | 0.714 | 0.786 | 0.78 |
HE | 0.449 | 0.323 | 0.401 | 0.525 | 0.74 | 0.729 | 0.622 | 0.742 | 0.745 | 0.72 | 0.708 | 0.718 |
FIS | 0.064 | -0.163 | 0.072 | 0.289 | 0.176 | 0.111 | 0.202 | 0.104 | 0.188 | 0.008 | -0.111 | -0.088 |
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N | 76 | 84 | 84 | 80 | 64 | 108 | 28 | 44 | 56 | 68 | 70 | 66 |
1 | 0 | 0 | 0 | 0 | 0.016 | 0 | 0 | 0 | 0.161 | 0.088 | 0 | 0.045 |
2 | 0 | 0 | 0 | 0 | 0.047 | 0.019 | 0.036 | 0.182 | 0.054 | 0 | 0.029 | 0.03 |
3 | 1 | 1 | 1 | 0.988 | 0.906 | 0.981 | 0.964 | 0.818 | 0.75 | 0.868 | 0.714 | 0.682 |
4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.018 | 0.044 | 0.257 | 0.242 |
5 | 0 | 0 | 0 | 0.013 | 0.031 | 0 | 0 | 0 | 0.018 | 0 | 0 | 0 |
HO | 0 | 0 | 0 | 0.025 | 0.188 | 0.037 | 0.071 | 0.364 | 0.429 | 0.206 | 0.371 | 0.576 |
HE | 0 | 0 | 0 | 0.025 | 0.178 | 0.037 | 0.071 | 0.304 | 0.416 | 0.241 | 0.429 | 0.481 |
FIS | NA | NA | NA | 0.000 | -0.054 | -0.010 | 0.000 | -0.200 | -0.032 | 0.148 | 0.136 | -0.202 |
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Mean HO | 0.226 | 0.208 | 0.212 | 0.249 | 0.39 | 0.432 | 0.372 | 0.572 | 0.495 | 0.473 | 0.54 | 0.563 |
HO SD | 0.212 | 0.191 | 0.192 | 0.195 | 0.212 | 0.342 | 0.262 | 0.18 | 0.098 | 0.255 | 0.218 | 0.224 |
Mean HE | 0.297 | 0.182 | 0.235 | 0.311 | 0.507 | 0.482 | 0.403 | 0.582 | 0.549 | 0.574 | 0.632 | 0.644 |
HE SD | 0.257 | 0.165 | 0.209 | 0.258 | 0.293 | 0.386 | 0.292 | 0.241 | 0.174 | 0.289 | 0.177 | 0.142 |
FIS | 0.241 | -0.148 | 0.098 | 0.200 | 0.235 | 0.105 | 0.078 | 0.018 | 0.100 | 0.178 | 0.148 | 0.168 |
Significant genetic differentiation (
Pop2 | Pop3 | Pop4 | Pop5 | Pop6 | Pop7 | Pop8 | Pop9 | Pop10 | Pop11 | Pop12 | |
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Pop1 | n.s. | * | n.s. | * | * | * | * | * | * | * | * |
Pop2 | n.s. | * | * | * | * | * | * | * | * | * | |
Pop3 | n.s. | * | * | * | * | * | * | * | * | ||
Pop4 | * | * | * | * | * | * | * | * | |||
Pop5 | n.s. | * | n.s. | * | * | * | * | ||||
Pop6 | n.s. | n.s. | * | * | * | * | |||||
Pop7 | n.s. | * | * | * | n.s. | ||||||
Pop8 | * | * | * | * | |||||||
Pop9 | * | * | * | ||||||||
Pop10 | * | n.s. | |||||||||
Pop11 | n.s. |
Spatial autocorrelation analysis of genetic variation at three allozyme loci (multi- and single-locus analysis) in the
Locus | Distance class [km] | ||||
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0–6 | 6–12 | 12–18 | 18–24 | 24–30 | |
Pairs of data | 16 | 20 | 14 | 11 | 7 |
all loci | 0.086 (- ***) | 0.171 (n.s.) | 0.196 (n.s.) | 0.206 (n.s.) | 0.326 (+ **) |
EST-X | 0.236 (- *) | 0.411 (n.s.) | 0.536 (n.s.) | 0.657 (n.s.) | 0.982 (+ **) |
GPI | 0.094 (- **) | 0.307 (n.s.) | 0.346 (n.s.) | 0.285 (n.s.) | 0.384 (n.s.) |
MPI | 0.014 (- *) | 0.015 (n.s.) | 0.030 (n.s.) | 0.033 (n.s.) | 0.062 (+ **) |
The shape of the aedeagus tip is not only used for taxonomic distinction between different carabids, but also for a justification of species rank (
The existence of a hybrid zone without strong selection pressure (since we found no deviations from Hardy-Weinberg equilibrium after correcting for multiple tests) despite strong differences in the shape of the aedeagus of both forms has consequences for taxonomy within the genus
The strong differentiation of the endophallus in some species of the subgenus
Numerous questions that are important for a better understanding of the investigated hybrid zone are still unanswered, e.g. if the contribution to gene flow into the hybrid zone is the same for both sexes or if the diffusion rate of markers differs. These and others can only be studied when further molecular, both mitochondrial and nuclear markers are analysed. mtDNA analysis, which is presently being conducted at our institute, may enable us to further estimate evolutionary divergence time between the two investigated subspecies with the help of phylogenetic analysis. The results of the present study reveal that
We thank Ingo Sponheuer for sampling some populations and laboratory work. Helpful comments on the manuscript provided by two anonymous reviewers are gratefully acknowledged.
Gel and staining recipes and electrophoresis conditions used in this study. (doi:
Copyright notice: This dataset is made available under the Open Database License (